Method of degrading trinitrotoluene

ABSTRACT

A method of eluting trinitrotoluene (TNT) from soil using a dispersant from bacterial intra-amoebic isolate 1s, ATCC 75229.

The United States Government has rights in this invention pursuant to contract no. DE-AC05-84OR21400 between the United States Department of Energy and Martin Marietta Energy Systems, Inc. The invention was funded by

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of application Ser. No. 08/168,603 filed on Dec. 16, 1993, now U.S. Pat. No. 5,449,618, which is a continuation-in-part of application Ser. No. 08/011,841, filed on Feb. 1, 1993, now U.S. Pat. No. 5,314,821, issued on May 24, 1994, entitled Amoeba/Bacteria Consortia and Uses for Degrading Wastes and Contaminants, the entire disclosure of which is incorporated herein by reference. Application Ser. No. 08/011,841 is a continuation of application Ser. No. 07/693,998, filed on Apr. 26, 1991, now abandoned.

FIELD OF THE INVENTION

The present invention relates to methods of biological degradation of napalm and trinitrotoluene (TNT), and more particularly to such methods which utilize intra-amoebic isolates. Napalm as used herein, is defined as napalm B, a mixture of gasoline, benzene, and polystyrene. The original, obsolete napalm formula, containing napthenic and palmitic acids, has not been tested.

BACKGROUND OF THE INVENTION

The end of the "cold war" has brought about the need for the reduction or elimination of many weapons stockpiles. Among those are explosives and incendiaries which, for the sake of protecting the environment, can no longer be burned. Therefore there is a need for alternative methods for eliminating explosives and incendiaries, in particular, waste napalm and TNT.

OBJECTS OF THE INVENTION

Accordingly, it is an object of the present invention to provide a new and improved method of biological degradation of napalm.

It is another object of the present invention to provide a new and improved method of biological degradation of TNT.

Further and other objects of the present invention will become apparent from the description contained herein.

SUMMARY OF THE INVENTION

In accordance with one aspect of the present invention, the foregoing and other objects are achieved by a method of degrading napalm which involves the steps of: deriving a dispersant from an intra-amoebic isolate essentially identical to American Type Culture Collection Deposit Number 75529; contacting napalm with the dispersant to produce partially degraded napalm; and, contacting the partially degraded napalm with at least one of intra-amoebic isolates essentially identical to American Type Culture Collection Deposit Numbers 75526 and 75527 to further degrade the partially degraded napalm.

In accordance with another aspect of the present invention, a method of eluting TNT from TNT contaminated soil involves the steps of: deriving a dispersant from an intra-amoebic isolate essentially identical to American Type Culture Collection Deposit Number 75529; loading TNT contaminated soil into a column; and, eluting the column with the dispersant to elute the TNT from the TNT contaminated soil.

In accordance with a further aspect of the present invention, a method of degrading TNT involves the steps of: providing an intra-amoebic isolate essentially identical to American Type Culture Collection Deposit Number 75528; and, contacting TNT with the intra-amoebic isolate to degrade the TNT.

DETAILED DESCRIPTION OF THE INVENTION

Amoeba/bacteria consortium 46, ATCC Deposit Reference No. 40908, was found to contain certain useful bacteria, designated as intra-amoebic isolates 13, NAP-1, 1S and CR-1. Isolate 1S produces a biodispersant which separates the components of naplam, and also elutes TNT bound to soil. Isolates NAP-1 and 13, in combination with the biodispersant produced from isolate 1S, degrade and disperse the polystyrene component of napalm. Isolate CR-1 degrades TNT in a manner which renders it undetectable by standard analysis methods.

The method of deriving a dispersant and using it to separate components of napalm is described in general terms as follows: Isolate 1S is usually grown on culture plates containing a solid medium. A typical solid medium suitable for this purpose comprises 15 g/L Bacto Tryptone (Pancreatic Digest of Casein), 5 g/L Bacto Soytone (Papaic Digest of Soybean Meal), 5 g/L Sodium Chloride and 15 g/L Bacto Agar. Small amounts of freshly grown bacteria (typically 1-3 day old cultures) are spread on the surface of the solid medium and incubated at a temperature of about 15° C. to about 40° C., preferably about 30° C., for several days (usually about 3-5 days) in an aerobic environment. When growth is sufficiently heavy, the bacteria are harvested, washed by centrifugation (usually about three times) with normal saline (about 8.5 g/L NaCl), diluted (to about 10 ml for each culture plate used) with normal saline, and autoclaved (typically for about 15 minutes at 121° C. at 15 psi). The autoclaved solution is allowed to cool, and is preferably filtered through a 0.2 μm nucleopore filter to remove extraneous membranes and other bacterial debris. The biodispersant solution thus derived is preferably stored in a sterile container. The biodispersant solution is preferably diluted 1:10 with sterile distilled water for use in separation of napalm components.

Napalm typically composed of polystyrene, leaded gasoline, and benzene, is degraded as follows: Napalm, preferably in its liquid form, is added to the diluted biodispersant solution. The mixture should generally contain about 5% to about 25%, preferably about 10%, napalm. Upon thorough mixing, an emulsifying effect is observed in the mixture, indicating suspension of the polystyrene component of the napalm.

To further degrade the mixture, a supernatant bacterial preparation containing about 10³ /ml to about 10⁸ /ml, usually about 10⁵ /ml, isolate 13 and/or isolate NAP-1, preferably both, is added to the emulsified mixture in a ratio of about 3:1 to about 1:3, preferably about 1:1. The mixture is then incubated at a temperature of about 15° C. to about 40° C., preferably about 30° C., until the polystyrene component of the napalm has dissolved, usually at least 24 hours. Marked increases in breakdown products of polystyrene, indicative of its degradation, will be evident, as seen in Table 1.

EXAMPLE I

A dispersant was derived from Isolate 1S as described hereinabove, and was mixed with napalm as described hereinabove to produce an emulsified mixture containing a suspension of the polystyrene component of the napalm. Results are shown in Table 1.

EXAMPLE II

Isolate 13 and isolate NAP-1 were grown separately on Trypticase Soy Agar at 30° C. for 3 days and harvested dry. The bacteria were prepared and used as described hereinabove to degrade the mixture produced in Example I. Results are shown in Table 1.

                  TABLE 1                                                          ______________________________________                                         Effect of Isolates 13 and Nap-1 on Volatile Aromatic                           Components of Napalm                                                                    Water   Biodis-   Biodispersant +                                              Control persant   Isolates 13, NAP-1                                  ______________________________________                                         Benzene in:                                                                    Polystyrene.sup.a                                                                         37,000,000                                                                               28,000,000                                                                               38,000,000                                      Supernate.sup.b                                                                           140,000   320,000   110,000                                         Ethyl Benzene in:                                                              Polystyrene                                                                               80,000    80,000    150,000                                         Supernate  420       620       150,000                                         Toluene in:                                                                    Polystyrene                                                                               81,000    91,000    130,000                                         Supernate  1,800     2,400     210,000                                         Total xylene in:                                                               Polystyrene                                                                               180,000   150,000   280,000                                         Supernate  1,200     820       260,000                                         ______________________________________                                          .sup.a μg/kg (semi solid portion of mixture)                                .sup.b μg/L (liquid portion of mixture)                               

A general, simple method of treating soil contaminated with TNT is described as follows. Soil contaminated with TNT is loaded into a vessel, usually a column, for contacting the soil with a biodispersant. An aqueous solution containing biodispersant derived from isolate 1S (as described hereinabove) is added and allowed to percolate down through the soil and elute the TNT and metabolic byproducts (metabolites) thereof. The solution may be forced through the soil, the soil may be pretreated, and/or other steps known to the skilled artisan may be taken to increase the efficiency of the process. The process may be carried out at about room temperature, but that is not a particularly critical factor.

EXAMPLE III

10 grams of contaminated soil were loaded into two columns, 7 in. in height with a diameter of 2.0 inches, each column having a Whatman #1 filter pad in the bottom thereof to retain the soil. One column was eluted with 100 ml distilled, deionized water, and the other column was eluted with 100 ml of the above described dispersant solution. The flow rate through the soil was slow, requiring 12 hours for all the liquid to pass through the columns. The eluate and soil for both the control and test columns were collected and analyzed for TNT and metabolites after acetonitrile extraction. No metabolites were detected. The results are shown in Table 2, indicating a significant reduction in detectable TNT in the soil eluted with the biodispersant solution.

                  TABLE 2                                                          ______________________________________                                         Desorption of TNT from Soil Using Biodispersants                               from Isolate 1S                                                                        VA Site (soil)                                                                          NJ Site (soil)                                                                             NJ Site (eluate)                                  ______________________________________                                         Water Control                                                                            5,900 μg/g                                                                             370 μg/g 15,750 μg/L                                10%       1,900 μg/g                                                                              5.3 μg/g                                                                                1,100 μg/L                                Biodispersant                                                                  ______________________________________                                    

A method of degrading TNT using isolate CR-1 is as follows. A solution is prepared containing a mineral salts solution usually NATE, and about 20 mg/L to about 200 mg/L, usually about 50 mg/L, TNT. Isolate CR-1 is grown on a suitable medium, usually Trypticase Soy Agar, for generally 1-2 days at about 15° C. to about 40° C., usually about 30° C., and harvested dry. Isolate CR-1 is added to the TNT solution at a concentration of about 10³ /ml to about 10⁹ /ml, usually about 10⁶ /ml, followed by incubation at about 15° C. to about 40° C., usually about 30° C., to degrade the TNT.

A typical NATE solution is generally prepared using the following materials:

Solution A (10× stock)

10 g/L MgSO₄.7H₂ O

2 g/L CaCl₂

10 g/L KNO₃

1 g/L NH₄ Cl

Solution B (100× stock)

5 mg/L CuSO₄. 5H₂ O

1 mg/L H₃ BO₃

1 mg/L MnSO₄ or 0.76 mg/L MNSO₄.1 H₂ O

7 mg/L ZnSO₄

1 mg/L MoO₃

1 mg/L CoCl₂.6H₂ O

Phosphate Buffer

8.5 g KH₂ PO₄ and 6.5 g K₂ HPO₄ in 300 ml

Iron Chloride

0.027 g/100 ml FeCl₃

To prepare a typical NATE media, 100 ml of solution B is added to 1 liter of solution A, resulting in a 10× NATE solution which can be autoclaved (sterilized). After cooling, the sterilized 10× NATE solution is diluted 1:10 with sterile water to obtain a 1× NATE solution. To 1 liter of the 1× NATE solution is added 20 ml filter sterilized phosphate buffer and add 10 ml of filter sterilized FeCl₃ to obtain the finished NATE media.

EXAMPLE IV

TNT was dissolved in water and diluted 1:1 with NATE media in a test bottle, resulting in a solution containing about 50 mg/L TNT. Isolate CR-1 was grown on Trypticase Soy Agar for 1-2 days at 30° C. and then harvested dry. Isolate CR-1 was then added to the TNT/NATE solution at a concentration of 10⁶ /ml, followed by incubation at 30° C. until the solution had attained a stable yellow-orange color. A control for this experiment was similar to the test, with the organisms killed by autoclaving prior to addition to the test bottle. Each bottle was analyzed by high pressure liquid chromatography (HPLC) and showed a marked decrease in TNT levels by the live bacteria. Small quantities of 4-amino-2,6-dinitrotoluene (4-ADNT), one of the metabolites associated with the degradation of TNT, were detected as indicated in Table 3.

                  TABLE 3                                                          ______________________________________                                         CONCENTRATION OF TNT AND ITS METABOLITES                                       AFTER INCUBATION WITH BACTERIA ISOLATE CR-1                                    Isolate      TNT, mg/L  4-ADNT, mg/L                                           ______________________________________                                         CR-1         36         0                                                      (5 minutes)                                                                    CR-1         10         0.3                                                    (24 hours)                                                                     Control      48         0                                                      (24 hours)                                                                     ______________________________________                                    

EXAMPLE V

Additional experiments were then undertaken using a similar protocol as previously mentioned except that the saturated TNT solution now contained ¹⁴ C-labeled TNT. Most of the ¹⁴ C-TNT was associated with the cell pellet. Further analysis of the cell pellet showed no detectable TNT present. TNT metabolites were detected in small amounts in the test bottles. Results are shown in Tables 4 and 5.

                  TABLE 4                                                          ______________________________________                                         % .sup.14 C-TNT per fraction                                                   EXPERIMENT   Cell Pellet  CO.sub.2                                                                              Soluble                                       ______________________________________                                         1            70           1      29                                            2            66           2      32                                            Control       6           4      90                                            ______________________________________                                    

                  TABLE 5                                                          ______________________________________                                         % TNT and Metabolites in Cell Pellet                                           EXPERIMENT   TNT        2-ADNT   4-ADNT                                        ______________________________________                                         1            0.0        0.009    0.04                                          2            0.0        0.005    0.05                                          Control      0.0        0.000    0.00                                          ______________________________________                                    

Deposit of Microorganisms

The applicants, in accordance with the provisions of the Budapest treaty on the international recognition of the deposit of microorganisms for the purposes of patent procedure under the Budapest Treaty, did deposit samples of Isolate NAP-1, Isolate 13, Isolate CR-1, and Isolate 1S with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md. 20852, U.S.A. on Aug. 20, 1993 and assigned ATCC deposit reference Numbers 75526, 75527, 75528, and 75529, respectively. Each culture is hereby irrevocably and without restriction or condition released to the public upon the issuance of letters patent herefor.

While there has been shown and described what are at present considered the preferred embodiments of the invention, it will be obvious to those skilled in the art that various changes and modifications can be made therein without departing from the scope of the inventions defined by the appended claims. 

What is claimed is:
 1. A method of eluting trinitrotoluene from trinitrotoluene contaminated soil comprising the steps of:a. autoclaving a bacterium selected from the group consisting of American Type Culture Collection Deposit Number 75529 and a mutant of said bacterium possessing all the identifying characteristics of said bacterium, to derive a dispersant solution therefrom; b. loading trinitrotoluene contaminated soil into a column; and, c. eluting said column with said dispersant solution in amounts and under conditions suitable to elute said trinitrotoluene from said trinitrotoluene contaminated soil. 